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Open Access Research article

The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein

Jolanda MP Liefhebber, Simone Punt, Willy JM Spaan and Hans C van Leeuwen*

Author Affiliations

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

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BMC Cell Biology 2010, 11:33  doi:10.1186/1471-2121-11-33

Published: 14 May 2010

Additional files

Additional file 1:

Localization of GLT25D1-HA compared to internally Myc-tagged GLT25D1. Huh7 cells were co-transfected with full length GLT25D1-HA (GLT25D1 FL-HA) and internally Myc-tagged full length GLT25D1 (GLT25D1 FL Myc-int.). 24 h after transfection the cells were subjected to immunofluorescence analysis. GLT25D1 FL-HA and GLT25D1 FL Myc-int. were detected by antibodies against HA (green) and Myc (red), respectively. Pearson correlation of GLT25D1 FL-HA and GLT25D1 FL Myc-int. is 0.83.

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Additional file 2:

GLT25D1 is a luminal ER protein. After 24 h, Huh7 cells transfected with GLT25D1 FL-HA were subjected to sucrose density gradient centrifugation. Cell lysates were loaded under a sucrose gradient from 10-80% w/v. Fractions were taken from top (fraction 1) to bottom (fraction 12) and separated by SDS-PAGE, followed by immunoblot analysis for Calnexin and PDI (protein disulfide isomerase). GLT25D1 FL-HA was visualized using an antibody against HA-epitope.

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Additional file 3:

GLT25D1 peptide coverage. Huh7 cell lysates were subjected to two-dimensional polyacrylamide gel electrophoresis, followed by silverstaining. Spots, corresponding to Mw 72 kDa and isoelectric point ~7 were digested with trypsin and analysed by ESI tandem MS. The obtained peptide sequences from GLT25D1 are shown in bold in the primary sequence of the protein.

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Additional file 4:

Sequence alignment of human GLT25D1, LH3(PLOD3) and close homologues. Alignment was generated using the ClustalX program. DXD motif is indicated (red box). Yellow indicates putative glycosyltransferase folds. FeII_Oxy indicates a predicted Fe(II)-dependent oxygenase superfamily. Numbers indicate amino acid position.

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